PHYTOTHERAPY RESEARCH Phytother. Res. 25: 530535 (2011) Published online 13 September 2010 in Wiley Online Library (wileyonlinelibrary.

com) DOI: 10.1002/ptr.3290

The Effect of Carvacrol on Muscarinic Receptors of Guinea-pig Tracheal Chains

M. H. Boskabady,1* Z. Jafari2 and I. Pouraboli2

ptr_3290

530..535

1 Department of Physiology and Pharmaceutical Research Centre, Medical School Mashhad University of Medical Sciences, Mashhad, Iran 2 Department of Biology, Bahonar University, Kerman, Iran

The effects of three concentrations of carvacrol, the constituent of Zataria multiora Boiss (a monoterpenoid phenol, C10H14O) and 10 nM atropine on muscarinic receptors were tested on: non-incubated (n = 7), incubated tracheal chains with propranolol and chlorpheniramine (n = 6) and incubated with propranolol (n = 5). The EC50 of all three concentrations of carvacrol in incubated tissues with propranolol and chlorpheniramine was signicantly greater than those of incubated tissues with propranolol and non-incubated trachea (p < 0.05 to p < 0.001). The EC50 of two higher concentrations of carvacrol (0.2 and 0.4 mg/mL) in incubated tissues with propranolol was also signicantly greater than those of non-incubated trachea (p < 0.01 to p < 0.001). The maximum response in the presence of all concentrations of carvacrol in non-incubated and incubated tissues with propranolol and chlorpheniramine and those of its two higher concentrations (0.2 and 0.4 mg/mL) in incubated tissues with propranolol were lower than saline (p < 0.05 to p < 0.001). There were parallel rightward shifts in the concentrationresponse curves in the presence of all concentrations of carvacrol in non-incubated and incubated tissues with propranolol and its lower concentration in incubated tissues with propranolol and chlorpheniramine. These results indicated an inhibitory effect of carvacrol on muscarinic receptors. A b-adrenoceptor stimulatory effect was also suggested for carvacrol. Copyright 2010 John Wiley & Sons, Ltd.
Keywords: muscarinic receptor; inhibitory effect; trachea; guinea-pig; carvacrol; Zataria multiora Boiss.

INTRODUCTION Carvacrol or cymophenol, C6H3CH3(OH)(C3H7) is the constituent of several medicinal plants including Zataria multiora Boiss (ESCOP, 1997), Thymus vulgaris (ESCOP, 1997), Carum copticum (Ballba et al., 1973) and Satureja hortensis (Paskov and DrianovskaNoninska, 1954; Radonic and Milos, 2003). For all plants containing carvacrol, a therapeutic effect on respiratory disease was described (ESCOP, 1997; Avesina, 1985; Zargari, 1992). In addition, all plants containing carvacrol showed a relaxant effect on different smooth muscles including trachea (Gharib Naseri, 2003; Reiter and Brandt, 1985; Boskabady et al., 2006; Boskabady et al., 2007; Boskabady et al., 1998; Hajhashemi et al., 2000; Boskabady et al., 2007). Another study showed that the relaxant effect of essential oil from Carum copticum on tracheal chains is due mainly to its fraction 2, which is suggested to be carvacrol (Boskabady et al., 2003). In addition, the relaxant effect of carvacrol on tracheal chains was also observed in another study (Boskabady and Jandaghi, 2003).
* Correspondence to: Dr M. H. Boskabady, Department of Physiology, Medical School, Mashhad University of Medical Science, Mashhad, Post Code 9177948564, Iran. E-mail: boskabadymh@mums.ac.ir; mhboskabady@hotmail.com

To examine one possible mechanism for the relaxant effect of carvacrol on smooth muscle, in the present study, the inhibitory effect of carvacrol on muscarinic receptors was examined on tracheal chains of guinea-pigs. MATERIALS AND METHODS Tissue preparations. Male Dunkin-Hartley guinea-pigs (400700 g) were killed by a blow on the neck and the trachea were removed. Each trachea was cut into 10 rings (each containing 23 cartilaginous rings). All the rings were then cut open opposite the trachealis muscle, and sutured together to form a tracheal chain (Holroyde, 1986). The tissue was then suspended in a 10 mL organ bath (organ bath 61300, Bio Science Palmer-Washington, Sheerness, Kent, UK) containing Krebs-Henseleit solution with the following composition (mm): NaCl 120, NaHCO3 25, MgSO4 0.5, KH2PO4 1.2, KCl 4.72, CaCl2 2.5 and dextrose 11. The Krebs solution was maintained at 37C and gassed with 95% O2 and 5% CO2. Tissue was suspended under isotonic tension (1 g) and allowed to equilibrate for at least 1 h while it was washed with Krebs solution every 15 min. The study was approved by the Universitys Ethics Committee. The allowance number of the relevant ethical committee for the animal experiments is 85301.
Received 07 May 2010 Revised 04 August 2010 Accepted 05 August 2010

Copyright 2010 John Wiley & Sons, Ltd.

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Protocols. The inhibitory effect of carvacrol on muscarinic receptors was examined by producing the cumulative log concentrationresponse curve of methacholine hydrochloride (Sigma Chemical Ltd, UK) induced contraction of tracheal chains 10 min after the exposure of tissue to each of the following solutions: (1) 10 nm atropine maleate (Sigma Chemical Ltd UK, Catalogue No. C4915); (2) three different concentrations of carvacrol (Fluka, Italy, Catalogue No. C4915, purity 75%), (0.1, 0.2 and 0.4 mg/mL); (3) 0.2 mL saline. Consecutive concentrations of methacholine were added every 2 min (range 0.11000 mm); and the percentage of contraction due to each concentration in proportion to the maximum contraction, obtained in the presence of saline, was plotted against log concentration of methacholine. The effective concentration of methacholine causing 50% of maximum response (EC50) in each experiment was measured using the log concentrationresponse curve of the corresponding experiment. The shift of cumulative log concentrationresponse curves obtained in the presence of different concentrations of carvacrol and atropine were examined by comparing the EC50 obtained in the presence of each solution with that of saline. In addition, the maximum responses to methacholine obtained in the presence of different concentrations of carvacrol and atropine in all sets of experiments were compared with that of saline. To examine the parallel rightward shift, the slope of the methacholine-response curve of each experiment was measured and compared with that of saline. In experiments with parallel shift in methacholine-response curve, the concentration-ratio minus one (CR - 1) as an index of the competitive antagonism effect was calculated by the following equation:

obtained in the presence of carvacrol and atropine were compared with those obtained in the presence of saline and (CR - 1) obtained in the presence of carvacrol, with those obtained in the presence of atropine using a paired t-test. The comparisons of the data of different concentrations of carvacrol were performed using oneway analysis of variance (ANOVA) with Tukey-Kramer multiple plot test. The values of EC50, the slope, (CR 1), and maximum response obtained in three groups were compared using ANOVA. Signicance was accepted at p < 0.05.

RESULTS Shift in cumulative log concentrationresponse curves Cumulative log concentrationresponse curves of methacholine obtained in the presence of all concentrations of carvacrol and atropine showed a clear rightward shift compared with methacholine curves produced in the presence of saline in all three groups of experiments (Fig. 1).

Tracheal responsiveness (EC50) The EC50 methacholine obtained in the presence of atropine in all experimental conditions was signicantly higher than that of saline (p < 0.001 for groups 1 and 2 and p < 0.05 for group 3). The EC50 obtained in the presence of all concentrations of carvacrol in all three groups was also signicantly greater than those of saline (p < 0.05 to p < 0.001) (Table 1). The EC50 methacholine obtained in the presence of all three concentrations of carvacrol in group 2 was signicantly higher than those of groups 1 and 3 (p < 0.05 to p < 0.001) (Table 1). In addition, the EC50 methacholine obtained in the presence of two higher concentrations of carvacrol (0.2 and 0.4 mg/mL) in group 3 was signicantly greater than those of group 1 (p < 0.01 for both cases) (Table 1).

EC 50 obtained in the presence of effective solutions 1 EC 50 obtained in the presence of saline

To ensure the notion of competitive antagonism of carvacrol, the Schild plot was constructed by plotting log (CR - 1) against log carvacrol concentrations. The study was performed on incubated tracheal chains 30 min prior to the beginning and while obtaining the methacholine-response curve with three different experimental designs as follows: (1) non-incubated tracheal chains (group 1 experiments), (n = 7); (2) incubated tissues with 1 mm chlorpheniramine (Sigma Chemical Ltd UK) and 1 mm propranolol hydrochloride (Sigma Chemical Ltd UK), (group 2 experiments), to inhibit histamine (H1) and b-adrenoceptors respectively (n = 6); (3) incubated tissues with 1 mm propranolol hydrochloride (group 3 experiments), to inhibit b-adrenoceptors (n = 5). All the experiments were performed randomly with a 1 h resting period of tracheal chains between each two experiments while washing the tissues every 15 min with Krebs solution. The three groups were studied in three different series of tracheal chains. In all experiments contractions were measured using an isotonic transducer (Harvard APP Ltd, 50-6360 SINO 0210) and measured using a software by computer (Acer model no: G781) recording. Statistical analysis. All data were expressed as mean SEM. The EC50, the slope and the maximum response
Copyright 2010 John Wiley & Sons, Ltd.

Maximum response to methacholine The maximum responses to methacholine obtained in the presence of all concentrations of carvacrol in groups 1 and 2 and its higher concentration (0.4 mg/mL) in group 3 were signicantly lower than that of saline (p < 0.05 to p < 0.001). The maximum responses obtained in the presence of all concentrations of carvacrol in groups 2 and 3 were improved compared with group 1 (p < 0.05 to p < 0.001) (Table 2). The maximum response obtained in the presence of only higher concentrations of carvacrol in group 3 was also signicantly higher than those of group 2 (p < 0.05) (Table 2). Slope of methacholine-response curves The slopes of methacholine-response curves obtained in the presence of different concentrations of carvacrol in
Phytother. Res. 25: 530535 (2011)

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M. H. BOSKABADY ET AL.

Tracheal contractile response (% maximum effect)

110 100 90 80 70 60 50 40 30 20 10 0

Saline Atropine Carvacrol 0.01 Carvacrol 0.02 Carvacrol 0.04

obtained in the presence of a low concentration of carvacrol (0.1 mg/mL) were signicantly lower than that of atropine (p < 0.001 and p < 0.05, respectively) (Table 4). All values of (CR - 1) obtained in groups 2 and 3 were signicantly higher than those of group 1 (p < 0.05 to p < 0.001) (Table 4). However, there was no signicant difference in (CR - 1) between groups 2 and 3 (Table 4).

Correlations between values of EC50 and concentrations of the extract and carvacrol There were signicant positive correlations between the concentrations of carvacrol and the values of EC50 in all three groups of experiments (r = 0.618, 0.815, 0.830, and p < 0.005, p < 0.001, p < 0.001 for groups 1, 2 and 3, respectively).

(a)

110 100 90 80 70 60 50 40 30 20 10 0

Schild plot The slopes of the Schild plot for carvacrol were -0.777, -0.981 and -0.970 in groups 1, 2 and 3, respectively (Fig. 2).

(b)
100 90 80 70 60 50 40 30 20 10 0

DISCUSSION The relaxant effect of carvacrol on tracheal chains (bronchodilatory) seen in a previous study (Boskabady and Jandaghi, 2003) might have been produced due to several different mechanisms including an inhibitory effect on muscarinic receptors because the relaxant effect of inhibition of this receptor has been shown (Loenders et al., 1992). Therefore, to clarify one possible mechanism responsible for the observed relaxant effect seen for carvacrol on tracheal muscle (Boskabady and Jandaghi, 2003), its inhibitory effect was examined on muscarinic receptors of isolated guinea-pig tracheal preparations in this study. The parallel rightward shifts in the methacholine log concentrationresponse curves obtained in the presence of the different concentrations of carvacrol, greater EC50 but lower maximum contraction effect to methacholine compared with that of saline in group 1 experiments (non-incubated trachea) may indicate functional antagonism effect of carvacrol at muscarinic receptors of guinea-pig trachea (Loenders et al., 1992; Arunlakshana and Schild, 1959; Ariens, 1987). To evaluate the contribution of the b-adrenergic stimulatory and/or histamine (H1) blocking effect on the functional antagonism seen for carvacrol, at muscarinic receptors seen in group 1, the inhibitory effects of carvacrol on these receptors were also examined on incubated tracheal preparation with propranolol and chlorpheniramine in group 2. The parallel rightward shift in methacholine-response curves obtained in the presence of different concentrations of carvacrol compared with that of saline and the signicant improvement in maximum responses to methacholine and the increase in EC50 obtained in this group of experiments, relative to those of group 1 showed possible competitive antagonistic effects of carPhytother. Res. 25: 530535 (2011)

(c)

-7

Methacholine Concentration (Log M)

-6

-5

-4

-3

-2

Figure 1. Cumulative log concentrationresponse curves of metacholine induced contraction of guinea-pig tracheal chains, in the presence of saline, three concentrations of carvacrol and 10 nM atropine on in (a) non-incubated trachea (group 1, n = 7), (b) incubated trachea with 1 mM chlorpheniramine and 1 mM propranolol (group 2, n = 6) and (c) incubated trachea with 1 mM propranolol (group 3, n = 5).

all three groups were not signicantly different from those of saline except at its higher concentration (0.4 mg/mL) in group 1 (p < 0.05). The slopes obtained in the presence of only higher concentrations of carvacrol in group 2 were signicantly higher than those of group 1 (p < 0.05) (Table 3). Shift in methacholine concentrationresponse curves (CR-1) The values of (CR - 1) obtained in the presence of all concentrations of carvacrol in group 1 were signicantly lower than that of atropine (p < 0.001 for all cases) (Table 4). In groups 2 and 3, the values of (CR - 1)
Copyright 2010 John Wiley & Sons, Ltd.

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Table 1. EC50 (mM) of methacholine obtained in the presence of carvacrol, 10 nM atropine and saline in three sets of experiments
Group 2 vs Group 1 NS p < 0.001 p < 0.001 p < 0.001 NS Group 3 vs Group 1 NS NS p < 0.01 p < 0.01 NS Group 3 vs Group 2 NS p < 0.01 p < 0.01 p < 0.05 NS

Solution Saline Carvacrol

Concentration

Group 1 9.57 0.95 15.14 2.58a 20.00 2.58b 28.67 4.05b 83.86 8.37b

Group 2 8.08 0.69 39.00 4.04b 66.67 4.41b 89.17 8.49b 82.50 9.09b

Group 3 6.04 1.11 21.90 3.34a 45.40 4.38b 61.40 5.83b 60.10 13.88a

0.1 mg/mL 0.2 mg/mL 0.4 mg/mL

Atropine

Values are presented as mean SEM. Group 1, experiments on non-incubated tracheal chains (n = 7); Group 2, experiments on tracheal chains incubated with 1 mM chlorpheniramine, 1 mM propranolol (n = 6) and Group 3, experiments on incubated tracheal chains with 1 mM propranolol (n = 5). NS, non-signicant difference. Statistical comparison in EC50 between saline and other solutions. NS, non-signicant difference. a p < 0.05, b p < 0.001.

Table 2. Maximum response to metacholine obtained in the presence of carvacrol, 10 nM atropine and saline in the three sets of experiments
Group 2 vs Group 1 NS p < 0.05 p < 0.05 p < 0.05 NS Group 3 vs Group 1 NS p < 0.01 p < 0.01 p < 0.001 NS Group 3 vs Group 2 NS NS NS p < 0.05 NS

Solution Saline Carvacrol

Concentration

Group 1 100.00 54.33 53.75 33.73 95.24 0.00 5.85 7.21 4.70 2.55

Group 2 100.00 77.83 77.00 57.81 99.30 0.00 6.75 7.51 8.07 0.50

Group 3 100.00 91.62 89.80 84.20 95.48 0.00 3.8 3.53 3.81 1.67

0.1 mg/mL 0.2 mg/mL 0.4 mg/mL

Atropine For abbreviations see Table 1.

Table 3. Slope of methacholine Log concentration-response curves in the presence of carvacrol, 10 nM atropine and saline in three sets of experiments
Group 2 vs Group 1 NS NS NS p < 0.05 NS Group 3 vs Group 1 NS NS NS NS 1.12 0.35 Group 3 vs Group 2 NS NS NS NS NS

Solution Saline Carvacrol

Concentration

Group 1 0.97 0.86 0.84 0.76 0.77 0.16 0.13 0.26 0.12 0.11

Group 2 0.99 0.95 0.94 0.98 0.99 0.06 0.02 0.03 0.01 0.07

Group 3 0.86 1.11 1.031 0.91 0.03 0.13 0.1 0.12

0.1 mg/mL 0.2 mg/mL 0.4 mg/mL

Atropine For abbreviations see Table 1.

Table 4. Values of (CR-1) in the presence of carvacrol and 10 nM atropine in three sets of experiments
Group 2 vs Group 1 p < 0.001 p < 0.001 p < 0.001 NS Group 3 vs Group 1 p < 0.05 p < 0.01 p < 0.01 NS Group 3 vs Group 2 NS NS NS NS

Solution Carvacrol

Concentration 0.1 mg/mL 0.2 mg/mL 0.4 mg/mL

Group 1 0.739 0.29b 1.11 0.27b 2.02 0.27b 8.14 1.06

Group 2 3.96 0.55b 7.68 1.12 NS 10.41 1.27 NS 9.31 0.83

Group 3 2.89 7.43 10.41 9.90 0.52a 1.40 NS 1.94 NS 2.12

Atropine

For abbreviations see Table 1. Statistical comparison in (CR-1) between chlorpheniramine and other solutions. NS, non-signicant difference, a p < 0.05, b p < 0.001.

vacrol on muscarinic receptors. The data from group 2 may also indicate an inhibitory effect on histamine (H1) receptors and/or adrenergic stimulatory effects for carvacrol. The similar values of (CR - 1) obtained in the presence of the concentrations of carvacrol, in group 2 compared with that of atropine indicates a comparable antagonistic effect of carvacrol relative to atropine at the concentrations used.
Copyright 2010 John Wiley & Sons, Ltd.

Although improvement in maximum responses was obtained in the presence of different concentrations of carvacrol in group 2 compared with those of group 1, there were still signicant differences between the maximum responses obtained in the presence of all concentrations of carvacrol with those of saline. These results indicate small functional antagonistic effects of this substance at muscarinic receptors rather than a
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534
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2

M. H. BOSKABADY ET AL.

(a)
1.2

-0.4

-0.6

-0.8

-1.0

1.0

0.8

0.6

(b)
1.2

0.4

-0.4

-0.6

-0.8

-1.0

1.0

0.8

0.6

b-adrenergic stimulatory and/or a histamine (H1) receptor blocking effect. To investigate whether changes in the results observed in group 2 are due to a b-adrenergic stimulatory or a histamine (H1) receptor blocking effect, the effect of carvacrol was examined on tissues incubated with propranolol in group 3. The results of this group showed a non-signicant difference in the slopes obtained in the presence of all concentrations of carvacrol with that of saline.The maximum responses obtained in the presence of two lower concentrations of carvacrol also were not signicantly different to that of saline and were greater than those of group 2. The results of this group indicate a b-adrenergic stimulatory effect for the extract. The cause of the reduction in EC50 is perhaps due to an increase of the maximum response in this group. These results may indicate that the relaxant effect observed for carvacrol in our previous study (Boskabady and Jandaghi, 2003) is mediated by muscarinic receptor blocking the b- and/or adrenergic stimulatory effect of this substance. The signicant positive correlations between the effects of carvacrol and the concentration indicated concentration-dependent inhibitory effect of this substance on muscarinic receptor. In addition, the slopes of the Schild plot in groups 2 and 3 were very close to minus one (-1) supporting the notion of competitive antagonism of carvacrol on muscarinic receptors of guinea-pig tracheal chains (Loenders et al., 1992). In conclusion, the results of this study suggested a competitive antagonistic effect of carvacrol at muscarinic receptors. A beta-adrenergic stimulatory effect was also suggested for carvacrol.

Log (CR-1)

0.4

Acknowledgements
0.2

(c)

-0.4

-0.6

-0.8

-1.0

-Log carvacrol concentration

This study was supported nancially by the Research Department of Mashhad University of Medical Sciences.

Figure 2. The Schild plot, log (CR - 1) against log carvacrol concentrations in groups 1 (a), 2 (b) and 3 (c). The slopes of Schild plot were -0.777, -0.981 and -0.970 in groups 1, 2 and 3, respectively.

Conict of Interest
The authors have declared that there is no conict of interest.

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